Cell culture chamber for a cell culture system

ABSTRACT

A cell culture chamber ( 20 ) for a closed cell culture system serving to continuously supply different cells with liquid nutrient media, growth factors, gases or the like essentially consists of a membrane plate ( 1 ) with a membrane ( 2 ) for accommodating at least one cell culture and with a number of channels ( 4, 4′, 4″, 4 ′″) for supplying liquid, gassing and connecting sensors, of a transparent glass pane ( 3 ), which is placed on one side of the membrane plate ( 1 ) while provided for observing the inside of the cell culture chamber ( 20 ) from this side, and consists of a connecting plate ( 5 ), which is placed on the opposite side of the membrane plate ( 1 ) and which is provided with an incorporated, transparent glass pane ( 6 ) for illuminating the inside of the cell culture chamber ( 20 ) from this opposite side with the aid of an assigned lighting system.

The invention relates to a cell culture chamber for a closed cellculture system serving to continuously supply cells of the most diversetype with liquid nutrient media, growth factors, gases and the like.

Essentially cell culture means a culture which is prepared on the basisof individual cells that either result from parts of tissue, primarycultures, from cell lines or cell stems obtained by enzymatic, mechanicor chemical disintegration. For the cultivation of the cells normallyculture vessels made of plastic that are incubated in CO₂ incubators areused. This guarantees a constant temperature (e.g. 37° C.) and abuffering of the medium by means of a 5 to 10 percent CO₂ gassing.Oxygen supply is effected by simple diffusion. With the known equipment,co-cultivation and freely changeable incubation conditions are normallynot possible.

For microscopic observation or for specific examinations, the culturevessels have to be taken out of the relevant incubators, wherebyincubation is interrupted, the cells cool down and the test conditionsare no longer constant as a result.

The previously known equipment or systems for the cultivation of cells,however, do no longer meet the requirements of modern cell culturetechnology.

With regard to current main research areas in the pharmaceuticalindustry in particular, these being the fields of inflammation(rheumatics), the fight against cancer, cardiovascular diseases, Aids,apoptosis (programmed cell death) and blood coagulation, the developmentand the testing of suitable new active agents and drugs by means of acell culture system, which shall be designed in such way that enablesthe testing of substances and actions under almost in-vivo conditions,that means with an almost perfect mapping of complex, biological systemsbefore passing on to the clinical phases (tests on test persons) isindispensable.

Out of consideration for the above-mentioned situation, there is demandfor a method for simulating the progress of reactions within one orseveral organ system(s) (e.g. by means of a series connection of cellculture chambers with hepatocytes and other types of cells, testing fordegradation products and metabolites) in order to considerably minimize,on the one hand, the period of time that passes between theidentification of the action of a substance and drug approval and toenable the obtaining of necessary findings on the mechanism of action ofthe substance within a complex biological system before passing on tothe clinical test phase, on the other hand.

A similar situation is given, for example, in the area of cosmeticsindustry as well.

State-of-the-art technology includes, for example, multivalent cellculture systems (see DE 199 15 178 A1, for example), problem-adaptedcell culture systems for specific tasks (see WO 98/17822, for example)or methods for the replication of cell cultures (see WO 97/37001 forexample).

Furthermore it is known from WO 99/23206 that there is a method formixing a cell culture infected with varicella in cylindrical bottles,for example.

Finally a method and a device used to take up a cell culture are knownfrom EP 0 999 266 A1; these aim at creating the most homogenous possibleconditions for the molecular biological or genetic examination of cells.

Out of consideration for the situation in the field of modern cellculture technology described in the beginning, the target of the presentinvention is now to create a new cell culture chamber for a closed cellculture system serving to accommodate at least one cell culture, and thecontinuously supply in particular of different cells with liquidnutrient media, growth factors, gases and the like in the cell culturechamber is guaranteed, without cells of a culture having to be taken outof their habitual environment, while all cell cultures can bepermanently examined under the microscope without the gassing having tobe interrupted.

This task is solved with regard to a cell culture chamber for a closedcell culture system for the continuous supply of different cells withliquid nutrient media, growth factors, gases and the like according tothe invention in such way that essentially the cell culture chamber iscomposed of the following components:

-   -   a) a membrane plate with a membrane for accommodation of a least        one cell culture and with a number of channels for supplying        liquid, gassing and connecting sensors;    -   b) a transparent glass pane, which is placed on one side of the        membrane plate while provided for observing the inside of the        cell culture chamber from this side; and    -   c) a cover plate, which is placed on the other opposite side of        the membrane plate, which is provided with an incorporated,        transparent glass pane for illuminating the inside of the cell        culture chamber from the mentioned other side by means of an        assigned lighting system.

Preferably here the transparent glass pane on the membrane plate forobserving the inside of the cell culture chamber is fixed in the area ofthe underside of the membrane plate.

Even more preferably the cover plate forms a cell culture chamber capwith a fixed integrated transparent glass pane and the cell culturechamber cap is fixed on the upper side of the membrane plate in areleasable way.

According to a further embodiment of the invention, it is provided thatthe cell culture chamber cap as well as the underside of the membraneplate have a opening for the accommodation and fixation of therespective glass pane, in particular for fixation, which is notreleasable.

Preferentially the respective transparent glass pane is a sapphire glasspane.

According to a further embodiment of the invention it may further morebe provided that for the fixation of the membrane on the membrane platea retaining ring is placed, which with the aid of the cell culturechamber cap can be pressed on the periphery of the membrane so that thelatter can be fixed.

A further embodiment of the invention is that preferentially on the sideof the cell culture chamber cap facing the membrane plate a joint ringis provided, by which, when the cell culture chamber is closed, the cellculture prepared on the membrane is aseptically closed.

Another, preferential development of the invention consists of the factthat by an suitable compartmentalization of the cell culture chamber aconstant, continuous gassing is enabled via the respective assignedchannels with freely selectable concentrations of the most differenttypes of gases. In particular this has the advantage that the cellculture in the inside of the cell culture chamber can be observedwithout interrupting the gassing.

In addition, it is also possible that the membrane plate on its sideopposite the cell culture chamber cap can be fixed on an assignedretainer plate for introduction into the cell culture system and thisretainer plate is fitted with an integrated heating for the cell culturechamber. Preferentially this heating is an electrical heating.

If a direct co-cultivation shall be executed, it is especiallyadvantageous to use a gas-permeable biofoil as membrane, as explained inmore detail below.

Now in the following the invention will be explained in more detail byapplication examples and the following will be showed:

FIG. 1 shows by means of a diagram a top view of a cell culture chamber;

FIG. 2 shows a sectional view of a cell culture chamber according toline A-A in FIG. 1;

FIG. 3 shows the sectional view of the cell culture chamber according toFIG. 2 in an exploded view;

FIG. 3A shows, by means of a diagram, a side elevation of a membraneplate of the cell culture chamber; and

FIG. 4 shows by means of a diagram a complete, closed cell culturesystem, in which a predefined number of cell culture chambers is used.

According to the FIGS. 1, 2, 3 and 3A, a cell culture chamber 20 ismaterially composed of a membrane plate 1, in which a membrane 2, inparticular a gas-permeable biofoil is placed, which serves to theaccommodation of at least one cell culture. In the shown applicationengineering, the membrane 2 is placed in the lower area of the membraneplate 1, in particular securely fixed.

The membrane plate 1 also is fitted with a number of channels 4, 4′, 4″and 4′″, which run in the inside of the cell culture chamber 20, and ofwhich channel 4 serves to connect sensors, channel 4′ to supply liquidsand gas, channel 4″ to supply liquids and channel 4′″ to withdrawliquids or gas, as shown in more detail below in FIG. 3A. Thus the cellculture can be equally supplied from above as well as from below. By thespecial system of channels in the membrane plate 1 it is in particularguaranteed that the required conditions of incubation can be realized.

In the area of the underside of the membrane plate 1, a transparentglass pane 3 is placed for observing the inside of the cell culturechamber 20. Such observation is preferentially carried out from theunderside of the membrane plate 1 with the aid of a video camera withmicroscope adapter, as further explained below.

On the upper side of the membrane plate 1, a cell culture chamber cap 5is placed, which forms an upper cover plate and is integrated in atransparent glass pane 6 for a lighting of the inside the cell culturechamber 20. The cell culture chamber cap 5 is fixed in particular on theupper side of the membrane plate 1 and, preferentially with the aid ofscrews 9, is bolted to the membrane plate 1 in a releasable way.

The cell culture chamber cap 5 as well as also the underside of themembrane plate 1 are provided with an opening for accommodating andfixing the respective glass pane 6 resp. 3.

On the cell culture chamber cap 5, the glass pane 6 preferentiallycovers a round opening 13. In an appropriate way at the membrane plate 1the glass pane 3 forms a lower cover under membrane 2.

Preferentially the transparent glass panes 3 and 6 are each sapphireglass panes.

For fixation of the membrane 2 on the membrane plate 1, a retaining ring7 is provided, which—with the aid of the cell culture chamber cap 5—canbe pressed on the periphery of the membrane 2 in order to fix the latterin the cell culture chamber 20.

Further more a joint ring 8 is placed on the side of the cell culturechamber cap 5 facing the membrane plate 1. By means of this joint ring8, the cell culture prepared on the membrane 2 is aseptically closedwhen the cell culture chamber 20 is closed (see FIG. 2).

FIG. 3A shows, by means of a diagram, a side view of the membrane plate1 with the orifices of channel 4 serving to connecting sensors, channel4′ serving to supply liquid or gas, channel 4″ serving to supply liquidand channel 4′″ serving to withdraw liquid or gas provided there.

The orifices of the channels 4, 4′, 4″, 4′″ as shown in FIG. 3A for oneside S of the membrane plate 1, are identically provided on all threeother sides of the membrane plate 1.

FIG. 3A also shows the placement of the gas-permeable membrane 2 in theinside of the membrane plate 1 with the placement of the membrane 2being made in such a manner that a defined compartmentalization of thecell culture chamber 20 results from this, which enables a directco-cultivation of two cell cultures. At such a direct co-cultivation onboth sides of the membrane 2 a cell culture each of different type isplaced and in particular the cells of the first cell culture growing onthe one side of the membrane 2, i.e. on the apical side, are suppliedwith a first flow of media via channel 4′, whereas the cells of thesecond cell culture growing on the other side of the membrane 2, i.e. onthe basolateral side, are supplied with a flow of media, which differfrom the first one, via channel 4″. Thus the cells on the apical sideact as cover layer, while the cells on the basolateral side act asinternal cells.

Channel 4′ leading to the apical side may also serve to gassing, inparticular to a constant, continuous gassing with freely selectableconcentrations of the most different gases.

As already mentioned, channel 4 serves to connect sensors.

Finally channel 4′″ serves to withdraw liquids or gases from the apicalside of the membrane 2.

The components of the cell culture chamber 20 particularly are made fromappropriate stainless steel, for example from stainless steel 1.4435.

After fitting the membrane plate 1 in the clean room, the cell culturechamber cap is placed and bolted with the membrane plate 1 by means ofthe screws 9. The screws are short screws 9, which fix the cell culturechamber cap 5 on the membrane plate 1. Here the cell culture, which isaccommodated by the membrane plate 1, is simultaneously closed in anaseptic way with the aid of the joint ring.

In this condition, the cell culture chamber 20 is assembled with aretainer plate 10 of a cell culture system (see FIG. 4).

For this purpose in particular, the membrane plate 1 on its sideopposite to the cell culture chamber cap 5 is fixed on the retainingplate 10, which has a retainer bolt for adjustment. For the purpose offixing the membrane plate 1 on the retaining plate 10, relatively longscrews 12 are provided. Furthermore the retaining plate has anintegrated heating, preferably an electrical heating for the cellculture chamber 20, as explained in more detail below.

In the shown application example, the cell culture chamber 20 ismaterially designed in a rectangular, parallelepiped form and has asquare elevation. Naturally also other geometrical realizations areconceivable.

In the application example shown by means of the FIGS. 1-3A, theorifices of the channels 4, 4′, 4″ and 4′″ are provided on all foursides S of the membrane plate 1 in identical way. But also here otherarrangements for these channels, which materially have a cylindricalform, are conceivable. Other cross sections of the channels are alsoconceivable.

It has to be mentioned, however, that the retaining plate 10 shows, foreach cell culture chamber to be fixed on it, a medium circular opening14, the cross section of which corresponds to the opening 13 of the cellculture chamber cap 5 opposite. This middle opening 14 of the retainingplate 10 ensures the observation of the inside of the cell culturechamber 20 from below by means of a video camera with a microscopeadapter, as explained in more detail in FIG. 4.

FIG. 4 shows the application of the cell culture chambers 20 accordingto the invention in a closed cell culture system 30.

In this cell culture system 30 for example six cell culture chambers 20are placed as group A on the retaining plate 10, which by its integratedheating E guarantees for the incubation during the operating time of thecell culture system 30 constant temperatures within each of the cellculture chambers 20 of the cell culture chamber group A.

In particular by means of this heating E, an electrical heating of therespective cell culture chamber 20 is effected, by which a very accuratetemperature control is possible. In particular the heating E is designedin such a way that each individual cell culture chamber 20 of the cellculture chamber group A can be individually heated.

It is a special advantage of the cell culture system 30 that the heatingE can be controlled by means of an assigned software. For this purpose asystem consisting of infrared temperature measuring device 25 isinstalled above the cell culture chamber group A in such a way that arespective infrared temperature measuring device 25 is assigned to eachindividual cell culture chamber 20. The respective infrared temperaturemeasuring device 25 senses, by means of a infrared beam 25′, thetemperature prevailing in the cell culture and permanently signals therespective measurement result to a computer-controlled monitoring andcontrol system G, which materially consists of a data processing system37 and a monitor 36. The individual infrared temperature measuringdevice, 25 are connected to the monitoring and control system G via ajoint interconnection line 45. If the initially preset temperatures inthe cell culture chambers 20 of the cell culture chamber group A change,a control and/or adjustment of the heating E is automatically effectedby the monitoring and control system G, i.e. the temperature prevailingin the individual cell culture chamber 20 is permanently adjusted toachieve a constant temperature. Instead of by means of infraredtemperature measuring devices, the temperature control might also beeffected by means of other suitable temperature sensors.

By means of the software included in the monitoring and control systemG, on the other hand, it can be enabled that the temperatures in theindividual cell culture chambers 20 of the cell culture chamber group Aare freely adjustable and changeable over the entire duration of theexperiment, if this should be necessary due to certain reasons.

For the purpose of a permanent microscopic observation of the inside ofthe relevant cell culture chamber 20, a video system B with anaccordingly assigned microscope system is provided. This video system Bwill be explained in more detail in the following.

Below every individual cell culture chamber 20 of the cell culturechamber group A that includes a total of six cell culture chambers inthis application example, a video camera 22 with a microscope adapter22′ is fitted on a mechanically adjustable, mobile table, thereforethere is a total of six video cameras 22 with accessory microscopeadapters 22′. Thus one video camera 22 each with a microscope adapter22′ serves to observe one cell culture chamber 20 each. After theexperiment has been started and after meaningful areas in the cellculture contained in the relevant cell culture chamber 20 have beenidentified, an observation sector in the cell culture chamber 20 isdetermined. The mechanically adjustable mobile table 23 is moved to thisobservation sector then by means of adjusting screws (not represented),then the mobile table 23 is locked and the video system B remains in thesame position over the entire duration of the experiment as a result.Furthermore the definition setting at the relevant microscope adapter22′ is adjusted at the start of the test. This adjustment process on therelevant microscope adapter 22′ is carried out for all six cell culturechambers 20 and then remains unchanged until the experiment has beencompleted.

The video system B is preferably controlled via the software containedin the monitoring and control system G as well. Here every individualvideo camera 22 with microscope adapter 22′ is controlled in theprocess. This is carried out in particular in such a way that picturesof the relevant cell culture in the cell culture chamber 20 are taken atfreely selectable intervals (every minute, for example), a light source24 fitted above the relevant cell culture chamber 20 illuminating therelevant cell culture at the relevant point in time at which such arecording is made, so that a sufficient illumination of the inside ofthe cell culture chamber 20 is ensured for the video recordings. Whenthe video recording is completed, the control switches the relevantlight source 24 into a weak, dimmed-out standby state until the nextvideo recording is made. The light beam and/or light cone that isemitted by each of the light sources 24 and that enters inside therelevant cell culture chamber 20 through the respective sapphire glasspane 6 is marked 24′ in FIG. 1.

All light sources 24 are connected to the monitoring and control systemG via a joint connecting line 46.

Every single light beam/light cone 24′ illuminates the entire area ofthe cell culture contained in the relevant cell culture chamber 20.

The video system B is also connected to the monitoring and controlsystem G via a line 47; from the monitoring system, the line 47 isconnected to a junction point 48 to which the individual video cameras22 are connected via correspondingly assigned lines.

The video system B with microscope system as described above is only onethe possible models. Another possible embodiment of such a system forthe permanent observation of the inside of the cell culture chamberscomprises a single observation system, consisting of a video camera anda microscope adapter, is installed on a mobile table; this mobile tablemoves to the six cell culture chambers 20 of the cell culture chambergroup A at freely selectable intervals. The adjustment of theobservation system is carried out for the individual cell culture at thestart of the test, this means preferably after meaningful areas havebeen identified in the relevant cell culture, by means of the respectivesoftware included in the monitoring and control system G, this meansthat the six target positions of the moving table on which theobservation system has been mounted are programmed by means of therespective computer program. On account of the mechanic tolerances ofthe moving table, however, it is necessary to include an area that islarger than the area inside the individual cell culture chamber to beobserved.

The software now serves to define the area to be observed within thislarger area. The software is able to record and to recognize contours,this means that the contours and the configuration of the cells isrecognized when the table moves in the direction of a cell culturechamber again and an initially defined observation area is recorded.

This observation system that has been explained last is not representedin detail in the drawings, but the individual cell culture chambers 20are also illuminated by means of the light sources 24, as it has alreadybeen explained in details above.

Furthermore the cell culture system 30 represented in FIG. 4 is equippedwith a dosage system C for liquids (e.g. liquid nutrient media and thelike) that is fitted with e.g. four liquid storage tanks 31 with oneassigned liquid take-off line 31′ each; these liquid take-off lines 31′constitute a group of lines 32. This group of lines 32 is, on the otherhand, connected to a pump system 29 through which the different cellculture chambers 20 of the cell culture chamber group A are suppliedwith freely selectable liquids that are contained in the liquid tanks31.

The pump system 29, on the other hand, is connected to a multi-valvemodule 30′ via a line 33. The liquids are supplied to the cell culturechamber group A from the multi-valve module 30′ via sterile hose linesystems 27 and 28; these liquids are passed on from the individual cellculture chambers 20 in a flexible manner. The liquid supply as well asthe withdrawal and passing on of liquids is carried out via sterile hosesystems that are installed with standard hose connecting elements anddistributors at the start of a test; this means that they are connectedto corresponding channels in the membrane plate 1 of a relevant cellculture chamber 20. Here the connection of the standard hose elements(not represented in detail in the drawings) with the assigned channelsof the membrane plate is adjusted in such a way that sterility isguaranteed.

For reasons of flexibility, the types of liquids, the directions offlow, the distribution of liquids and their flow volumes can be changedand/or controlled during an experiment; this is preferably controlled bythe computer-controlled monitoring and control system G. For thispurpose, the pump system 29 is connected to the monitoring and controlsystem G via a connecting line 38 and the multi-valve module 30′ via aconnecting line 40.

Therefore the dosage system C of the cell culture system 30 enables youto supply the cell culture chamber group A with a variety of differentliquids.

Furthermore the cell culture system 30 is equipped with a gassing systemD for a variety of different gases. This gassing system D also serves togas the different cell culture chambers 20 of the cell culture chambergroup A with a variety of different gases, e.g. air, O₂, N₂, CO₂. Fromthe gassing system D, the gas is supplied to the cell culture chambergroup A via a sterile hose line 26. Also here the gases can be passed onfrom the different cell culture chambers 20 by means of the respectivelyassigned channels 4′ and 4′″ (see FIG. 3A) in a flexible manner.

The gas is altogether supplied withdrawn and passed on via sterile tubesthat are installed by means of standard hose connecting elements anddistributors at the start of an experiment.

The connections of the hose connecting elements with the correspondinglyassigned channels 4′ and 4′″ of the membrane plate 1 are adjusted insuch a way so that sterility is ensured.

With the gassing system D as well, the types of gases, the directions offlow, the gas distribution as well as the gassing concentration can bechanged and/or controlled during an experiment for reasons offlexibility. For this purpose, the gassing system D, on the other hand,is connected to the monitoring and control system G that contains therelevant software for controlling the gassing system D via a connectingline 39.

Finally the cell culture system 30 further includes a monitoring systemF with predefined sensor modules 34. By means of this monitoring systemF, the relevant parameters in the relevant cell culture chamber 20 ofthe cell culture chamber group A can be measured, measured permanentlyin particular, using accordingly assigned sensors, for the entireduration of a test, these parameters being, for example, pH value,glucose, lactate, oxygen, electric potential, etc. For this purpose, themonitoring system F is connected to the individual cell culture chambers20 of the cell culture chamber group A of the cell culture system 30 viaa line 41, via a junction point 42 and from there via further lines 43und 44 and accordingly assigned branch lines.

The parameters measured by the sensors (not shown) are transmitted bythe monitoring system F via a line 35 to the computer-controlledmonitoring and control system G for further processing. As alreadyexplained above in the FIGS. 1 to 3A, the cell culture chamber 20 isequipped with at least one channel 4 for sensor connection. The sensorsand the relevant assigned channel 4 of the membrane plate 1 are adjustedto each other in such a way so as to ensure sterility.

In summary, the closed cell culture system 30 equipped with cell culturechambers 20 according to the invention is able to simulate highlycomplex biological processes in real time and under almost in-vivoconditions, i.e. as in living organisms.

1. Cell culture chamber (20) for a closed cell culture system serving tocontinuously supply different cells with liquid nutrient media, growthfactors, gases and the like, characterized by the fact that the cellculture chamber (20) is materially composed of the following components:a) a membrane plate (1) with a membrane (2) serving to accommodate atleast one cell culture and equipped with a number of channels (4,4′, 4″,4′″) for supplying liquid, gassing and sensor connection; b) atransparent glass pane (3) which is placed on one side of the membraneplate (1) while provided for observing the inside of the cell culturechamber (20) from the side mentioned above; and c) a cover plate (5)which is placed on the opposite side of the membrane plate (1) and whichis provided with an incorporated, transparent glass pane (6) forilluminating the inside of the cell culture chamber (20) from thisopposite side with the aid of an assigned lighting system.
 2. Cellculture chamber according to claim 1, characterized by the fact that thetransparent glass pane (3) is fixed on the membrane plate (1) in thearea of the underside of the membrane plate (1) for the purpose ofobserving the inside of the cell culture chamber (20).
 3. Cell culturechamber according to claim 1, characterized by the fact that the coverplate (5) forms a cell culture chamber cap with a securely integratedtransparent glass pane (6), such cell culture chamber cap being fixed ina releasable manner on the upper side of the membrane plate (1).
 4. Cellculture chamber according to claim 1, characterized by the fact that thecell culture chamber cap (5) as well as the underside of the membraneplate (1) show an opening for the accommodation and fixation of therespective glass pane (6 resp. 3).
 5. Cell culture chamber according toclaim 1, characterized by the fact that the glass pane (6, 3) is asapphire glass pane.
 6. Cell culture chamber according to claim 1,characterized by the fact that, for the fixation of the membrane (2) onthe membrane plate (1), a retaining ring (7) is provided, which can,with the aid of the cell culture chamber cap (5), be pressed on theperiphery of the membrane (2) so that the latter can be fixed.
 7. Cellculture chamber according to claim 1, characterized by the fact that, onthe side of the cell culture chamber cap (5) facing the membrane plate(1), a joint ring (8) is provided, by which the cell culture prepared onthe membrane (2) is aseptically closed when the cell culture chamber(20) is closed.
 8. Cell culture chamber according to claim 1,characterized by the fact that a constant, continuous gassing is enabledby means of a suitable compartmentalization of the cell culture chamber(20) via the respectively assigned channels (4′, 4′″) with freelyselectable concentrations of the most various gases.
 9. Cell culturechamber according to claim 1, characterized by the fact that themembrane plate (1) can be fixed at its side opposite the cell culturechamber cap (5) on an assigned retainer plate (10) for integration inthe cell culture system, this retaining plate (10) being equipped withan integrated heating for the cell culture chamber (20).
 10. Cellculture chamber according to claim 9, characterized by the fact that theheating is an electrical heating.
 11. Cell culture chamber according toclaim 1, characterized by the fact that the membrane (2) is agas-permeable biofoil.